JPH0894511A - Measuring method for protein quantity using oscillator sensor - Google Patents

Abstract

PURPOSE: To perform high sensitive measurement by measuring the change quantity of an oscillator resulting from a produced precipitation through the addition of a protein coagulation reagent to a sample as making a protein coagulation reinforcement substance coexist with a precipitation reaction so as to quantitatively determine a very little quantity of protein. CONSTITUTION: Food, nutrient drinking water, serum, and urine are to be objects as samples. Trichloroacetic acid, sulfosalicylic acid, or the like is used as a protein coagulation reagent. A nonionic surfactant such as triton X-100, sarcosinate PN, or the like, or an amphoteric surfactant such as alkylamino ethyl glycine, or the like is used as a protein coagulation reinforcement substance. The protein coagulation reagent is added to the sample, and the change quantity of an oscillator resulting from a produced precipitation as making the protein coagulation reinforcement substance coexist with a precipitation reaction so as to make it possible to quantitatively determine very little quantity of protein.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein amount measuring method using a vibrator sensor which is useful in foods, clinical tests and the like.

[0002]

2. Description of the Related Art Conventional methods for quantifying protein content include the Burette method, Kjeldahl method, trichloroacetic acid / ponso-method.
A colorimetric method such as S method, a method using a refractometer, a turbidimetric method using a protein coagulation reagent, etc. are generally used. Of these, except for the refractometer method, the operation is complicated, and although the refractometer method is simple, errors are likely to occur and the sensitivity is low, so that it is difficult to measure samples other than serum.

[0003]

[Problems to be Solved by the Invention] There is a strong demand for a simple, rapid and highly sensitive method for measuring the amount of protein.

[0004]

The present invention is based on the finding that the resonance frequency of a crystal oscillator changes due to precipitation caused by a protein and a protein coagulation reagent. It was also found that the coexistence of a protein coagulation enhancer enhances the resonance frequency change and enables measurement of an extremely small amount of protein.

In the present invention, samples include food, nutritional drinking water, serum, urine, spinal fluid, and body fluid. Examples of protein coagulation reagents include trichloroacetic acid, sulfosalicylic acid, sulfosalicylic acid-sodium sulfate, and β-naphthalenesulfonic acid. Although the present invention has been carried out using a crystal oscillator, it can be applied in principle to a piezoelectric oscillator, a crystal Lamb wave device, a surface acoustic wave device, and the like. Gold, silver, palladium, etc. are suitable for the electrodes of the crystal unit, but aluminum, platinum, etc. may be used.

Triton X is used as a protein coagulation enhancer.
-100, nonionic surfactants such as sarcosinate PN, and amphoteric surfactants such as alkylaminoethylglycine. These enhancers may be added together with the protein coagulant, or may be added after a certain period of time after the protein coagulant is added.

A sample having a protein concentration of 1 to 300 mg / dl is diluted as it is or with water, and a sample having a higher concentration than this is diluted with water. Saline may be used for dilution. It is preferable that the pH of the alkaline sample be adjusted to neutral or lower with hydrochloric acid or the like. An appropriate amount of the sample thus pretreated is taken, and then 3 to 8 times, preferably 5 times, the amount of the coagulation reagent having a concentration of 3 g / l is added, and the mixture is vigorously stirred or stirred. 10 to
100 μl is injected onto a crystal oscillator and the change in resonance frequency is measured.

The protein coagulation enhancer has a coagulation reagent content of 0.0
It is added so as to be 2 to 0.5%, preferably 0.05 to 0.2%, or a considerable amount is added after stirring the coagulation reagent, and further stirred vigorously. Specific examples of the present invention will be described below.

[0009]

Example 1 (1) Preparation of Solution for Protein Level Calibration Curve Transparent, non-hemolytic human serum was dissolved in physiological saline, and a protein level standard solution with a protein concentration of 1, 10, 50, 100, 200 mg / dl was prepared. Prepared. (2) Quantification of protein amount 0.50 ml of a protein amount standard solution was placed in a test tube, and 3 g / dl trichloroacetic acid-0.1% Triton X-10 was added to this.
Add 2.5 ml of 0 aqueous solution and stir vigorously. Immediately, 50 μl of the solution was taken and injected into a crystal oscillator sensor (metal electrode, 9 MHz) manufactured by Mutual Yakuhin Co., Ltd., and a change in resonance frequency was measured. A large change occurred after injection, and a calibration curve was prepared with the frequency change for 3 minutes as the vertical axis. It is shown in FIG.
Similar results were obtained with the 0.01% Tego solution.

[0010]

Example 2 In Example 1, an experiment was conducted using 3 g / dl sulfosartyl-7% sodium sulfate instead of trichloroacetic acid. The calibration curve is shown in FIG.

[0011]

EFFECTS OF THE INVENTION A simple protein quantification is possible. Highly sensitive measurement can be performed by using a protein coagulation enhancer.

[Brief description of drawings]

FIG. 1 is a calibration curve of proteins obtained by the methods of Example 1 and Example 2.

[Explanation of symbols]

 ●-● Calibration curve of Example 1 ▲ Black square ▼ − ▲ Black square ▼ Calibration curve of Example 2

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[Procedure amendment]

[Submission date] February 15, 1995

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be amended] Title of invention

[Correction method] Change

[Correction content]

[Title of Invention] Protein amount measuring method using vibrator sensor

Claims (2)

[Claims] 1. A method for quantifying the amount of protein by adding a protein coagulation reagent to a sample and measuring the amount of change in the oscillator due to the generated precipitate. 2. The protein amount measuring method according to claim 1, wherein a protein coagulation enhancer is allowed to coexist in the precipitation reaction to quantify a trace amount of protein.